Plasma Membrane Localization of Apoptotic Caspases for Non-apoptotic Functions.

Caspases are best characterized for their function in apoptosis. However, they also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), where caspases release mitogens for compensatory proliferation independently of their apoptotic role. Here, we report that the unconventional myosin, Myo1D, which is known for its involvement in left/right development, is an important mediator of AiP in Drosophila. Mechanistically, Myo1D translocates the initiator caspase Dronc to the basal side of the plasma membrane of epithelial cells where Dronc promotes the activation of the NADPH-oxidase Duox for reactive oxygen species generation and AiP in a non-apoptotic manner. We propose that the basal side of the plasma membrane constitutes a non-apoptotic compartment for caspases. Finally, Myo1D promotes tumor growth and invasiveness of the neoplastic scrib RasV12 model. Together, we identified a new function of Myo1D for AiP and tumorigenesis, and reveal a mechanism by which cells sequester apoptotic caspases in a non-apoptotic compartment at the plasma membrane.

Killers creating new life: caspases drive apoptosis-induced proliferation in tissue repair and disease.

Apoptosis is a carefully orchestrated and tightly controlled form of cell death, conserved across metazoans. As the executioners of apoptotic cell death, cysteine-dependent aspartate-directed proteases (caspases) are critical drivers of this cellular disassembly. Early studies of genetically programmed cell death demonstrated that the selective activation of caspases induces apoptosis and the precise elimination of excess cells, thereby sculpting structures and refining tissues. However, over the past decade there has been a fundamental shift in our understanding of the roles of caspases during cell death-a shift precipitated by the revelation that apoptotic cells actively engage with their surrounding environment throughout the death process, and caspases can trigger a myriad of signals, some of which drive concurrent cell proliferation regenerating damaged structures and building up lost tissues. This caspase-driven compensatory proliferation is referred to as apoptosis-induced proliferation (AiP). Diverse mechanisms of AiP have been found across species, ranging from planaria to mammals. In this review, we summarize the current knowledge of AiP and we highlight recent advances in the field including the involvement of reactive oxygen species and macrophage-like immune cells in one form of AiP, novel regulatory mechanisms affecting caspases during AiP, and emerging clinical data demonstrating the critical importance of AiP in cancer.

Extracellular Reactive Oxygen Species Drive Apoptosis-Induced Proliferation via Drosophila Macrophages.

Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Here we show that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. We propose that in an immortalized ("undead") model of AiP, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer.

The Sound of Silence: Signaling by Apoptotic Cells.

Apoptosis is a carefully choreographed process of cellular self-destruction in the absence of inflammation. During the death process, apoptotic cells actively communicate with their environment, signaling to both their immediate neighbors as well as distant sentinels. Some of these signals direct the anti-inflammatory immune response, instructing specific subsets of phagocytes to participate in the limited and careful clearance of dying cellular debris. These immunomodulatory signals can also regulate the activation state of the engulfing phagocytes. Other signals derived from apoptotic cells contribute to tissue growth control with the common goal of maintaining tissue integrity. Derangements in these growth control signals during prolonged apoptosis can lead to excessive cell loss or proliferation. Here, we highlight some of the most intriguing signals produced by apoptotic cells during the course of normal development as well as during physiological disturbances such as atherosclerosis and cancer.

Detecting caspase activity in Drosophila larval imaginal discs.

Caspases are a highly specialized class of cell death proteases. Since they are synthesized as inactive full-length zymogens, activation--at least of effector caspases and to some extent also of initiator caspases-requires a proteolytic cleavage event, generating a large and a small subunit, two of each forming the active caspase. The proteolytic cleavage event generates neo-epitopes at both the C-terminus of the large subunit and the N-terminus of the small subunit. The cleaved Caspase-3 (CC3) antibody was raised against the neo-epitope of the large subunit and thus detects only cleaved, but not full-length, Caspase-3. Although raised against human cleaved Caspase-3, the CC3 antibody cross-reacts in other species and detects cleaved caspases, most notably DrICE and Dcp-1, in Drosophila. This protocol describes the procedure for use of the CC3 antibody to detect caspase activity in larval imaginal discs in Drosophila.

Genetic models of apoptosis-induced proliferation decipher activation of JNK and identify a requirement of EGFR signaling for tissue regenerative responses in Drosophila.

Recent work in several model organisms has revealed that apoptotic cells are able to stimulate neighboring surviving cells to undergo additional proliferation, a phenomenon termed apoptosis-induced proliferation. This process depends critically on apoptotic caspases such as Dronc, the Caspase-9 ortholog in Drosophila, and may have important implications for tumorigenesis. While it is known that Dronc can induce the activity of Jun N-terminal kinase (JNK) for apoptosis-induced proliferation, the mechanistic details of this activation are largely unknown. It is also controversial if JNK activity occurs in dying or in surviving cells. Signaling molecules of the Wnt and BMP families have been implicated in apoptosis-induced proliferation, but it is unclear if they are the only ones. To address these questions, we have developed an efficient assay for screening and identification of genes that regulate or mediate apoptosis-induced proliferation. We have identified a subset of genes acting upstream of JNK activity including Rho1. We also demonstrate that JNK activation occurs both in apoptotic cells as well as in neighboring surviving cells. In a genetic screen, we identified signaling by the EGFR pathway as important for apoptosis-induced proliferation acting downstream of JNK signaling. These data underscore the importance of genetic screening and promise an improved understanding of the mechanisms of apoptosis-induced proliferation.

Epitaxial relationships between uric acid crystals and mineral surfaces: a factor in urinary stone formation.

Uric acid (C5H4N4O3) is one of the final products of purine metabolism. Its concentration balance is maintained in the kidneys, but compromised kidney function can result in its crystallization either in the renal tract or in the interstitial fluid of joints. In physiological deposits, crystalline uric acid is most frequently found either in a protonated state (anhydrous or dihydrate phases) or as a deprotonated urate ion (sodium or ammonium salts). Often these precipitates are found in association with a number of mineral phases (e.g., calcium oxalates, calcium phosphates, and magnesium phosphates). Their frequent and common coexistence suggests that synergistic relationships between these crystalline phases may exist. A comprehensive list of different heterogeneous uric acid/uric acid and uric acid/mineral interfaces that are epitaxially matched was generated with the lattice-matching program EpiCalc. Two hundred twenty-five coincident epitaxial matches and four commensurate epitaxial matches were identified using this screening procedure.